C1q/Tumor Necrosis Factor-Related Protein-9 Is a Novel Vasculoprotective Cytokine That Restores High Glucose-Suppressed Endothelial Progenitor Cell Functions by Activating the Endothelial Nitric Oxide Synthase.

BACKGROUND: This study investigated whether gCTRP9 (globular C1q/tumor necrosis factor-related protein-9) could restore high-glucose (HG)-suppressed endothelial progenitor cell (EPC) functions by activating the endothelial nitric oxide synthase (eNOS). METHODS AND RESULTS: EPCs were treated with HG (25 mmol/L) and gCTRP9. Migration, adhesion, and tube formation assays were performed. Adiponectin receptor 1, adiponectin receptor 2, and N-cadherin expression and AMP-activated protein kinase, protein kinase B, and eNOS phosphorylation were measured by Western blotting. eNOS activity was determined using nitrite production measurement. In vivo reendothelialization and EPC homing assays were performed using Evans blue and immunofluorescence in mice. Treatment with gCTRP9 at physiological levels enhanced migration, adhesion, and tube formation of EPCs. gCTRP9 upregulated the phosphorylation of AMP-activated protein kinase, protein kinase B, and eNOS and increased nitrite production in a concentration-dependent manner. Exposure of EPCs to HG-attenuated EPC functions induced cellular senescence and decreased eNOS activity and nitric oxide synthesis; the effects of HG were reversed by gCTRP9. Protein kinase B knockdown inhibited eNOS phosphorylation but did not affect gCTRP9-induced AMP-activated protein kinase phosphorylation. HG impaired N-cadherin expression, but treatment with gCTRP9 restored N-cadherin expression after HG stimulation. gCTRP9 restored HG-impaired EPC functions through both adiponectin receptor 1 and N-cadherin-mediated AMP-activated protein kinase /protein kinase B/eNOS signaling. Nude mice that received EPCs treated with gCTRP9 under HG medium showed a significant enhancement of the reendothelialization capacity compared with those with EPCs incubated under HG conditions. CONCLUSIONS: CTRP9 promotes EPC migration, adhesion, and tube formation and restores these functions under HG conditions through eNOS-mediated signaling mechanisms. Therefore, CTRP9 modulation could eventually be used for vascular healing after injury.

Identification of C1q/TNF-related protein 4 as a novel appetite-regulating peptide that reduces food intake in Siberian sturgeon (Acipenser baerii).

Emerging findings point to a role for C1q/TNF-related protein 4 (CTRP4) in feeding in mammals. However, it remains unknown whether CTRP4 regulates feeding in fish. This study aimed to determine the feeding regulation function of CTRP4 in Siberian sturgeon (Acipenser baerii). In this study, the Siberian sturgeon ctrp4 (Abctrp4) gene was cloned, and Abctrp4 mRNA was shown to be highly expressed in the hypothalamus. In the hypothalamus, Abctrp4 mRNA decreased during fasting and reversed after refeeding. Subsequently, we obtained the AbCTRP4 recombinant protein by prokaryotic expression and optimized the expression and purification conditions. Siberian sturgeon (81.28 ± 14.75 g) were injected intraperitoneally using 30, 100, and 300 ng/g Body weight (BW) AbCTRP4 to investigate its effect on feeding. The results showed that 30, 100, and 300 ng/g BW of the AbCTRP4 significantly reduced the cumulative food intake of Siberian sturgeon at 1, 3, and 6 h. Finally, to investigate the potential mechanism of CTRP4 feeding inhibition, 300 ng/g BW AbCTRP4 was injected intraperitoneally. The findings demonstrated that AbCTRP4 treatment for 1 h significantly promoted the mRNA levels of anorexigenic peptides (pomc, cart, and leptin) while suppressing the mRNA abundances of orexigenic peptides (npy and agrp).In addition, the jak2/stat3 pathway in the hypothalamus was significantly activated after 1 h of AbCTRP4 treatment. In conclusion., this study confirms the anorexigenic effect of CTRP4 in Siberian sturgeon.

CTRP9 prevents atherosclerosis progression through changing autophagic status of macrophages by activating USP22 mediated-de-ubiquitination on Sirt1 in vitro.

BACKGROUND: Atherosclerosis (AS) is commonly regarded as a key driver accounted for the leading causes of morbidity and mortality among cardiovascular and cerebrovascular diseases. A growing body of evidence indicates that autophagy in macrophages involved in AS might be a potential therapeutic target. C1q/TNF-related protein 9 (CTRP9) has been proven to delay the progression of cardiovascular diseases. However, the relations between CTRP9 and Sirt1, as well as their effects on macrophages autophagy have not been fully explored. METHODS: Macrophages were differentiated from mononuclear cells collected from peripheral blood samples of healthy donors. The in vitro AS models were constructed by ox-LDL treatment. Cell viability was determined by CCK-8 assay. Immunofluorescence assay of LC3 was implemented for evaluating autophagy activity. Oil Red O staining was performed for lipid accumulation detection. ELISA, cholesterol concentration assay and cholesterol efflux analysis were conducted using commercial kits. Cycloheximide assay was implemented for revealing protein stability. RT-qPCR was used for mRNA expression detection, and western blotting was performed for protein level monitoring. RESULTS: CTRP9 attenuated impaired cell viability, autophagy inhibition and increased lipid accumulation induced by ox-LDL. Moreover, CTRP9 maintained Sirt1 protein level through enhancing its stability through de-ubiquitination, which was mediated by upregulated USP22 level. CRTP9 exerted its protective role in promoting autophagy and reducing lipid accumulation through the USP22/Sirt1 axis. CONCLUSION: Collectively, CTRP9 alleviates lipid accumulation and facilitated the macrophages autophagy by upregulating USP22 level and maintaining Sirt1 protein expression, thereby exerting a protective role in AS progression in vitro.

Responses of multifunctional immune complement component 1q (C1q) and apoptosis-related genes in Macrophthalmus japonicus tissues and human cells following exposure to environmental pollutants.

Apoptosis is a key defense process for multiple immune system functions, playing a central role in maintaining homeostasis and cell development. The purpose of this study was to evaluate the effects of environmental pollutant exposure on immune-related apoptotic pathways in crab tissues and human cells. To do this, we characterized the multifunctional immune complement component 1q (C1q) gene and analyzed C1q expression in Macrophthalmus japonicus crabs after exposure to di(2-ethylhexyl) phthalate (DEHP) or hexabromocyclododecanes (HBCDs). Moreover, the responses of apoptotic signal-related genes were observed in M. japonicus tissues and human cell lines (HEK293T and HCT116). C1q gene expression was downregulated in the gills and hepatopancreas of M. japonicus after exposure to DEHP or HBCD. Pollutant exposure also increased antioxidant enzyme activities and altered transcription of 15 apoptotic signaling genes in M. japonicus. However, patterns in apoptotic signaling in response to these pollutants differed in human cells. HBCD exposure generated an apoptotic signal (cleaved caspase-3) and inhibited cell growth in both cell lines, whereas DEHP exposure did not produce such a response. These results suggest that exposure to environmental pollutants induced different levels of immune-related apoptosis depending on the cell or tissue type and that this induction of apoptotic signaling may trigger an initiation of carcinogenesis in M. japonicus and in humans as consumers.

C1q/TNF-related protein 3 alleviates heart failure via attenuation of oxidative stress in myocardial infarction rats.

C1q-tumor necrosis factor-related protein 3 (CTRP3), an adipokine, has been reported to be closely related to cardiovascular diseases (CVDs). However, the effect of CTRP3 on heart failure (HF) remains dimness. This study was to explore the role of CTRP3 in HF and its potential interaction mechanism. Heart failure model was established by inducing ischemia myocardial infarction (MI) through ligation of the left anterior descending artery in Sprague-Dawley rats. Four weeks later, the rats were detected by transthoracic echocardiography and masson staining. Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cardiac troponin I (cTnI) levels, creatine kinase-MB (CK-MB) and oxidative stress levels were recorded. The level of CTRP3 was reduced in the cardiomyocytes (CMs) treated with oxygen-glucose deprivation (OGD) and in the heart of MI rats. CTRP3 overexpression alleviated cardiac dysfunction, attenuated the cardiac fibrosis, and inhibited the increases of ANP, BNP, cTnI and CK-MB in the serum of MI rats. The increases of ANP and BNP in the CMs, and the collagen I and collagen III in the cardiac fibroblasts (CFs) induced by OGD were inhibited by CTRP3 overexpression. The enhancement of oxidative stress in the heart of MI rats was inhibited by CTRP3 overexpression. These results indicated that overexpression of CTRP3 could improve cardiac function and the related cardiac fibrosis in MI-induced HF rats via inhibition of oxidative stress. Upregulation of CTRP3 may be a strategy for HF therapy in the future.

Astrocytic and microglial interleukin-1ß mediates complement C1q-triggered orofacial mechanical allodynia.

Glial cells, such as microglia and astrocytes, in the trigeminal spinal subnucleus caudalis (Vc) are activated after trigeminal nerve injury and interact with Vc neurons to contribute to orofacial neuropathic pain. Complement C1q released from microglia has been reported to activate astrocytes and causes orofacial mechanical allodynia. However, how C1q-induced phenotypic alterations in Vc astrocytes are involved in orofacial pain remains to be elucidated. Intracisternal administration of C1q caused mechanical allodynia in the whisker pad skin and concurrent significant upregulation of glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 in the Vc. Immunohistochemical analyses clarified that C1q induces a significant increase in the cytokine interleukin (IL)-1ß, predominantly in Vc astrocytes and partially in Vc microglia. The number of c-Fos-positive neurons in the Vc increased significantly in response to C1q. IL-1 receptor antagonist (IL-1Ra) was used to analyze the involvement of IL-1ß in C1q-induced mechanical allodynia. Intracisternal administration of IL-1Ra ameliorated C1q-induced orofacial mechanical allodynia. The present findings suggest that IL-1ß released from activated astrocytes and microglia in the Vc mediates C1q-induced orofacial pain.

Exenatide increases CTRP3 gene expression in adipose cells by inhibiting adipogenesis and induces apoptosis.

Considering the rapidly increasing prevalence of obesity worldwide, the number of weight control drugs is very few. Incretin-based therapies are currently being developed to achieve weight control, and Glucagon-Like Peptide-1 Receptor Agonists (GLP-1RA) are used in incretin-based therapies. This study aimed to investigate the cytotoxicity of exenatide, a GLP-1A, on 3T3-L1 adipocytes and the effect of exenatide on the expression of adipogenesis-related genes, insulin and glucose levels, and apoptosis. Cytotoxic activity of exenatide on 3T3-L1 adipocytes was determined by MTT method. Gene expression levels were determined by qPCR. Apoptosis studies were performed on the Muse Cell Analyzer. C1q/TNF-related protein-3 (CTRP3) expression levels were found to be higher in exenatide treated adipocyte cells than in control cells (p < 0.001). Adipocyte cells treated with exenatide were found to have lower PPAR-γ gene expression levels when compared to control adipocyte cells (p < 0.001). Intracellular insulin (p < 0.001) and glucose levels were higher in 3T3-L1 adipocytes treated with exenatide compared to control adipocyte cells. Total apoptosis increased approximately 1.5 times as a result of exenatide administration. The increase in CTRP3 gene expression, which is thought to be a new biomarker for obesity, and the decrease in PPAR-γ gene expression indicate that exenatide is a promising new pharmacotherapeutic agent in the treatment of obesity by regulating the expression of genes related to adipogenesis and lipogenesis and inducing apoptosis.

Bu Shen Yi Sui Capsule Promotes Myelin Repair by Modulating the Transformation of A1/A2 Reactive Astrocytes In Vivo and In Vitro.

Background/Aims. Multiple sclerosis (MS) is an autoimmune disorder that affects the central nervous system (CNS) primarily hallmarked by neuroinflammation and demyelination. The activation of astrocytes exerts double-edged sword effects, which perform an integral function in demyelination and remyelination. In this research, we examined the therapeutic effects of the Bu Shen Yi Sui capsule (BSYS), a traditional Chinese medicine prescription, in a cuprizone- (CPZ-) triggered demyelination model of MS (CPZ mice). This research intended to evaluate if BSYS might promote remyelination by shifting A1 astrocytes to A2 astrocytes. Methods. The effects of BSYS on astrocyte polarization and the potential mechanisms were explored in vitro and in vivo utilizing real-time quantitative reverse transcription PCR, immunofluorescence, and Western blotting. Histopathology, expression of inflammatory cytokines (IL-10, IL-1ß, and IL-6), growth factors (TGF-ß, BDNF), and motor coordination were assessed to verify the effects of BSYS (3.02 g/kg/d) on CPZ mice. In vitro, A1 astrocytes were induced by TNF-α (30 ng/mL), IL-1α (3 ng/mL), and C1q (400 ng/mL), following which the effect of BSYS-containing serum (concentration of 15%) on the transformation of A1/A2 reactive astrocytes was also evaluated. Results and Conclusions. BSYS treatment improved motor function in CPZ mice as assessed by rotarod tests. Intragastric administration of BSYS considerably lowered the proportion of A1 astrocytes, but the number of A2 astrocytes, MOG+, PLP+, CNPase+, and MBP+ cells was upregulated. Meanwhile, dysregulation of glutathione peroxidase, malondialdehyde, and superoxide dismutase was reversed in CPZ mice after treatment with BSYS. In addition, the lesion area and expression of proinflammatory cytokines were decreased and neuronal protection factors and anti-inflammatory cytokines were increased. In vitro, BSYS-containing serum suppressed the A1 astrocytic markers' expression and elevated the expression levels of A2 markers in primary astrocytes triggered by C1q, TNF-α, and IL-1α. Importantly, the miR-155/SOCS1 signaling pathway was involved in the modulation of the A1/A2 phenotype shift. Overall, this study demonstrated that BSYS has neuroprotective effects in myelin repair by modulating astrocyte polarization via the miR-155/SOCS1 pathway.

Deficiency of C1QL1 Reduced Murine Ovarian Follicle Reserve Through Intraovarian and Endocrine Control.

Ovarian aging is associated with depletion of the ovarian follicle reserve, which is the key determinant of fertility potential in females. In this study, we found that the small, secreted protein complement 1Q-like (C1QL1) is involved in the regulation of follicle depletion through intraovarian and endocrine control in a multidimensional collaborative manner. C1ql1 was detected to be conserved in the ovary and showed high transcript levels during folliculogenesis. Blockade of C1QL1 by IP and ovarian intrabursal injection of C1QL1 antiserum into prepubertal mice impaired folliculogenesis accompanied by reductions in body weight, fat mass, and intraovarian lipid accumulation. An elevation of circulating estradiol levels, reduction of hypothalamic KISS1 and GnRH expression, and a decrease in serum FSH levels were found in C1QL1-deficient mice. In C1QL1-deficient ovaries, many primordial follicles were recruited and developed into medium follicles but underwent atresia at the large follicle stages, which resulted in depletion of follicle reserve. Depletion of C1QL1 alleviated the inhibitory effect of C1QL1 on granulosa cell apoptosis and the stimulatory effect of C1QL1 on granulosa cell autophagy, which resulted in accumulation in the preantral and early antral follicles and an increase in the atretic follicles. The abnormal profile of endocrine hormones accelerated the intraovarian effect of C1QL1 deficiency and further led to depletion of ovarian reserve. Altogether, this study revealed the expression patterns and the mechanism of action of C1QL1 during folliculogenesis and demonstrated that deficiency of C1QL1 caused ovarian follicular depletion.

Amyloid beta acts synergistically as a pro-inflammatory cytokine.

The amyloid beta (Aß) peptide is believed to play a central role in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. However, the natural, evolutionarily selected functions of Aß are incompletely understood. Here, we report that nanomolar concentrations of Aß act synergistically with known cytokines to promote pro-inflammatory activation in primary human astrocytes (a cell type increasingly implicated in brain aging and AD). Using transcriptomics (RNA-seq), we show that Aß can directly substitute for the complement component C1q in a cytokine cocktail previously shown to induce astrocyte immune activation. Furthermore, we show that astrocytes synergistically activated by Aß have a transcriptional signature similar to neurotoxic "A1" astrocytes known to accumulate with age and in AD. Interestingly, we find that this biological action of Aß at low concentrations is distinct from the transcriptome changes induced by the high/supraphysiological doses of Aß often used in in vitro studies. Collectively, our results suggest an important, cytokine-like function for Aß and a novel mechanism by which it may directly contribute to the neuroinflammation associated with brain aging and AD.

Complement 1q protects MRL/lpr mice against lupus nephritis via inhibiting the nuclear factor-κB pathway.

Lupus nephritis (LN) is a kidney disorder that is a critical cause of mortality in patients with systemic lupus erythematosus. The present study aimed to explore the protective role of complement component 1q (C1q) on LN and the underlying mechanism involving the nuclear factor (NF)­κB singling pathway. MRL/lpr mice served as the LN mouse model, and pcDNA­C1q was injected into LN mice to determine the role of C1q. C1q mRNA expression was detected using reverse transcription­quantitative PCR. Urine protein and blood urea nitrogen (BUN) levels were measured, and the histological damage index was determined using H&E staining. ELISA was used to measure the levels of tumor necrosis factor­α (TNF­α), interleukin (IL)­1ß, IL­6, anti­C1q and anti­double stranded DNA (dsDNA). CD68­ and Ki67­positivity were detected using immunofluorescence, and NF­κB­related protein expression was examined using western blotting. C1q mRNA expression was downregulated in renal tissues of LN mice. Overexpression of C1q decreased urine protein, BUN levels and the histological damage index in LN mice. The levels of TNF­α, IL­1ß, IL­6, anti­C1q and anti­dsDNA in renal tissues of LN mice were also reduced after pcDNA­C1q treatment. Additionally, overexpression of C1q decreased the CD68­ and Ki67­positivity in glomeruli and attenuated the expression of NF­κB­related proteins. Phorbol 12­myristate 13­acetate, an NF­κB pathway activator, reversed the inhibitory effect of C1q on inflammation, macrophage infiltration and mesangial cell (MC) proliferation in renal tissues of LN mice. Thus, it was demonstrated that C1q ameliorated inflammation and macrophage infiltration and decreased MC proliferation in renal tissues of LN mice by inhibiting the NF-κB pathway.

Alcohol Increases Exosome Release from Microglia to Promote Complement C1q-Induced Cellular Death of Proopiomelanocortin Neurons in the Hypothalamus in a Rat Model of Fetal Alcohol Spectrum Disorders.

Microglia, a type of CNS immune cell, have been shown to contribute to ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing ß-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined whether the microglial extracellular vesicle exosome is involved in the ethanol-induced neuronal death of the ß-endorphin neuron. Extracellular vesicles were prepared from hypothalamic tissues collected from postnatal rats (both males and females) fed daily with 2.5 mg/kg ethanol or control milk formula for 5 d or from hypothalamic microglia cells obtained from postnatal rats, grown in cultures for several days, and then challenged with ethanol or vehicle for 24 h. Nanoparticle tracking analysis and transmission electron microscopy indicated that these vesicles had the size range and shape of exosomes. Ethanol treatments increased the number and the ß-endorphin neuronal killing activity of microglial exosomes both in vivo and in vitro Proteomics analyses of exosomes of cultured microglial cells identified a large number of proteins, including various complements, which were elevated following ethanol treatment. Proteomics data involving complements were reconfirmed using quantitative protein assays. Ethanol treatments also increased deposition of the complement protein C1q in ß-endorphin neuronal cells in both in vitro and in vivo systems. Recombinant C1q protein increased while C1q blockers reduced ethanol-induced C3a/b, C4, and membrane attack complex/C5b9 formations; ROS production; and ultimately cellular death of ß-endorphin neurons. These data suggest that the complement system involving C1q-C3-C4-membrane attack complex and ROS regulates exosome-mediated, ethanol-induced ß-endorphin neuronal death.SIGNIFICANCE STATEMENT Neurotoxic action of alcohol during the developmental period is recognized for its involvement in fetal alcohol spectrum disorders, but the lack of clear understanding of the mechanism of alcohol action has delayed the progress in therapeutic intervention of this disease. Proopiomelanocortin neurons known to regulate stress, energy homeostasis, and immune functions are reported to be killed by developmental alcohol exposure because of activation of microglial immune cells in the brain. While microglia are known to use extracellular vesicles to communicate with neurons for maintaining homeostasis, we show here that ethanol exposure during the developmental period hijacks this system to spread apoptotic factors, including complement protein C1q, to induce the membrane attack complex and reactive super-oxygen species for proopiomelanocortin neuronal killing.

The regulatory role of C1q on Helicobacter pylori-induced inflammatory cytokines secretion in THP-1 cells.

C1q, as a LAIR-1 ligand, maintains monocytes quiescence and possess immunosuppressive properties. To understand the roles and molecular mechanisms, C1q mediated inflammation cytokines and several pivotal proteins in THP-1 cells after H. pylori infection were detected. The results showed that the expression of IL-8, IL-10, LAIR-1, phosphorylated/total JNK, phosphorylated/total p38-MAPK, phosphorylated/total AKT and phosphorylated/total NF-κB were up-regulated significantly in THP-1 cells after H. pylori infection. There was significant upregulation in IL-10 concentration, phosphorylated/total p38-MAPK and phosphorylated/total AKT, and downregulation in phosphorylated/total JNK in non-H. pylori infected THP-1 cells pretreated with C1q. C1q was also able to increase IL-8 and IL-10 production, and reduce LAIR-1 and phosphorylated/total p38-MAPK expression in pretreatment-C1q THP-1 cells after H. pylori infection. These results together indicated that H. pylori might induce IL-8 and IL-10 production through JNK, p38-MAPK, PI3K/AKT and NF-κB signaling pathway. C1q manipulate LAIR-1 to regulation IL-8 and IL-10 secretion in THP-1 cells after H. pylori infection through the p38-MAPK signaling pathway. This information is helpful to further understand the role and mechanisms of C1q on inflammation cytokines secretion in monocytes after H. pylori infection.

Non-specific adsorption of complement proteins affects complement activation pathways of gold nanomaterials.

The complement system is a key humoral component of innate immunity, serving as the first line of defense against intruders, including foreign synthetic nanomaterials. Although gold nanomaterials (AuNMs) are widely used in nanomedicine, their immunological response is not well understood. Using AuNMs of three shapes commonly used in biomedical applications: spherical gold nanoparticles, gold nanostars and gold nanorods, we demonstrated that AuNMs activated whole complement system, leading to the formation of SC5b-9 complex. All three complement pathways were simultaneously activated by all the AuNMs. Recognition molecules of the complement system interacted with all AuNMs in vitro, except for l-ficolin, but the correlation between these interactions and corresponding complement pathway activation was only observed in the classical and alternative pathways. We also observed the mediating role of complement activation in cellular uptake of all AuNMs by human U937 promonocytic cells, which expresses complement receptors. Taken together, our results highlighted the potential immunological challenges for clinical applications of AuNMs that were often overlooked.

C1ql1/Ctrp14 and C1ql4/Ctrp11 promote angiogenesis of endothelial cells through activation of ERK1/2 signal pathway.

C1ql-like (C1QL)-1 and -4 proteins are encoded by homologous genes that are highly expressed in brain and adipose tissues. However, functional properties of C1QL proteins outside of the brain and adipocytes remain unknown. Here, we report that the globular domain of C1ql1/Ctrp14 and C1ql4/Ctrp11 proteins directly stimulate the angiogenesis of endothelial cells. In this study, soluble C1ql1/CTRP14 and C1ql4/Ctrp11 proteins, produced in prokaryote expression system, are co-cultured with human umbilical vein endothelium cells (HUVECs), which phenotype is identified with von Willebrand factor antibody. C1ql1/Ctrp14 and C1ql4/Ctrp11 promote the migration and capillary tube formation of HUVECs in a dose-dependent manner. During this process, phosphorylation of c-Raf, MEK1/2, ERK1/2, and p90RSK are activated by C1ql1/Ctrp14 and C1ql4/Ctrp11. MEK1/2 inhibitor, U0126, blocks C1ql1/Ctrp14-, and C1ql4/Ctrp11-induced capillary tube formation and cell migration. Moreover, the immunoreactivity of the receptor of C1QL1-C1QL4, brain-specific angiogenesis inhibitor 3 (BAI3), is detected in HUVECs, suggesting that BAI3 may mediate C1QL1/CTRP14- and C1QL4/CTRP11-induced angiogenesis. Meanwhile, C1ql1/Ctrp14 and C1ql4/Ctrp11 exposure also causes a stimulatory response of angiogenesis in chick yolk sac membrane. These data demonstrate that C1ql1/Ctrp14 and C1ql4/Ctrp11 stimulate the new blood vessel growth by activation of ERK1/2 signal pathway. The proangiogenic activity of C1ql1/Ctrp14 and C1ql4/Ctrp11 provides novel insights into the new opportunities for therapeutic intervention by targeting C1QLs in tumorigenesis, tissue regeneration, and recovery of ischemic heart disease.

Complimentary action: C1q increases ganglion cell survival in an in vitro model of retinal degeneration.

Using a previously described retinal explant culture system as an acute injury model, we here explore the role of C1q, the initiator of the classical complement pathway, in neuronal cell survival and retinal homeostasis. Full-thickness adult rat retinal explants were divided into four groups, receiving the following supplementation: C1q (50nM), C1-inhibitor (C1-inh; Berinert; 500mg/l), C1q+C1-inh, and no supplementation (culture controls). Explants were kept for 12h or 2days after which they were examined morphologically and with a panel of immunohistochemical markers. C1q supplementation protects ganglion cells from degeneration within the explant in vitro system. This effect is correlated to an attenuated endogenous production of C1q, and a quiesced gliotic response.

ADAM28 is expressed by epithelial cells in human normal tissues and protects from C1q-induced cell death.

ADAM28 (disintegrin and metalloproteinase 28), which was originally reported to be lymphocyte-specific, is over-expressed by carcinoma cells and plays a key role in cell proliferation and progression in human lung and breast carcinomas. We studied ADAM28 expression in human normal tissues and examined its biological function. By using antibodies specific to ADAM28, ADAM28 was immunolocalized mainly to epithelial cells in several tissues, including epididymis, bronchus and stomach, whereas lymphocytes in lymph nodes and spleen were negligibly immunostained. RT-PCR, immunoblotting and ELISA analyses confirmed the expression in these tissues, and low or negligible expression by lymphocytes was found in the lymph node and spleen. C1q was identified as a candidate ADAM28-binding protein from a human lung cDNA library by yeast two-hybrid system, and specific binding was demonstrated by binding assays, immunoprecipitation and surface plasmon resonance. C1q treatment of normal bronchial epithelial BEAS-2B and NHBE cells, both of which showed low-level expression of ADAM28, caused apoptosis through activation of p38 and caspase-3, and cell death with autophagy through accumulation of LC3-II and autophagosomes, respectively. C1q-induced cell death was attenuated by treatment of the cells with antibodies against the C1q receptor gC1qR/p33 or cC1qR/calreticulin. Treatment of C1q with recombinant ADAM28 prior to addition to culture media reduced C1q-induced cell death, and knockdown of ADAM28 using siRNAs increased cell death. These data demonstrate that ADAM28 is expressed by epithelial cells of several normal organs, and suggest that ADAM28 plays a role in cell survival by suppression of C1q-induced cytotoxicity in bronchial epithelial cells.

Complement protein C1q modulates neurite outgrowth in vitro and spinal cord axon regeneration in vivo.

Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI.

Human endogenous peptide p33 inhibits detrimental effects of LL-37 on osteoblast viability.

BACKGROUND AND OBJECTIVE: High levels of the antimicrobial peptide, LL-37, are detected in gingival crevicular fluid from patients with chronic periodontitis. LL-37 not only shows antimicrobial activity but also affects host-cell viability. The objective of the present study was to identify endogenous mechanisms that antagonize the detrimental effects of LL-37 on osteoblast viability, focusing on the human peptide p33 expressed on the surface of various cell types. MATERIAL AND METHODS: Human osteoblast-like MG63 cells and human hFOB1.19 osteoblasts were treated with or without LL-37 in the presence or absence of p33. Recombinant human p33 was expressed in an Escherichia coli expression system. Lactate dehydrogenase (LDH) was assessed using an enzymatic spectrophotometric assay. DNA synthesis was determined by measuring [(3) H]-thymidine incorporation. Cell number was assessed by counting cells in a Bürker chamber. Intracellular Ca(2+) was monitored by recording Fluo 4-AM fluorescence using a laser scanning confocal microscope. Cellular expression of p33 was determined by western blotting. RESULTS: LL-37 caused a concentration-dependent release of LDH from human osteoblasts, showing a half-maximal response value (EC50 ) of 4 µm and a rapid and sustained rise in the intracellular Ca(2+) concentration of osteoblasts, suggesting that LL-37 forms pores in the cell membrane. p33 (10 µm) inhibited the LL-37-induced LDH release and LL-37-evoked rise in intracellular Ca(2+) concentration, suggesting that p33 prevents LL-37-induced permeabilization of the cell membrane. Moreover, p33 blocked LL-37-induced attenuation of osteoblast numbers. Also, mucin antagonized, at concentrations representative for nonstimulated whole saliva, LL-37-evoked LDH release, whilst cationic endogenous polyamines had no impact on LL-37-induced LDH release from osteoblasts. CONCLUSIONS: The endogenous peptide p33 prevents LL-37-induced reduction of human osteoblast viability. Importantly, this mechanism may protect the osteoblasts from LL-37-induced cell damage in patients suffering from chronic periodontitis associated with high levels of LL-37 locally.

Complement protein C1q promotes macrophage anti-inflammatory M2-like polarization during the clearance of atherogenic lipoproteins.

OBJECTIVE: Innate immune protein C1q plays a dual role in the chronic inflammatory disease of atherosclerosis. Complement activation via C1q exacerbates pathology in the atherosclerotic lesion in later stages of the disease. However, in early stages of disease C1q is protective. We hypothesize that complement-independent activities of C1q are involved in reprogramming macrophage inflammatory polarization. METHODS: The influence of C1q on macrophage inflammatory responses during clearance of oxLDL was examined. Changes in cytokines at the gene and protein level were measured by quantitative PCR and ELISA assay. RESULTS: C1q modulated cytokine expression in Raw264.7 macrophages during ingestion of oxLDL. Levels of pro-inflammatory cytokines IL-1ß and IL-6 were downregulated by C1q, whereas levels of the anti-inflammatory cytokine IL-10 were increased. In addition, data from an NFκB-luciferase gene reporter assay suggest that C1q suppresses activation of NFκB during lipoprotein clearance in macrophages, providing one mechanism by which C1q downregulates pro-inflammatory cytokine production. CONCLUSIONS: C1q-polarization of macrophages toward an anti-inflammatory (M2-like) phenotype may be important in dampening inflammation in the early atherosclerotic lesion. Further investigation of molecular pathways targeted by C1q may provide novel therapeutic targets for this disease.